Ureases: Historical aspects, catalytic, and non-catalytic properties – A review

Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-containing enzyme produced by plants, fungi, and bacteria that catalyzes the hydrolysis of urea into ammonia and carbamate. Urease is of historical importance in Biochemistry as it was the first enzyme ever to be crystallized (1926). Finding nickel in urease’s active site (1975) was the first indication of a biological role for this metal. In this review, historical and structural features, kinetics aspects, activation of the metallocenter and inhibitors of the urea hydrolyzing activity of ureases are discussed. The review also deals with the non-enzymatic biological properties, whose discovery 40 years ago started a new chapter in the study of ureases. Well recognized as virulence factors due to the production of ammonia and alkalinization in diseases by urease-positive microorganisms, ureases have pro-inflammatory, endocytosis-inducing and neurotoxic activities that do not require ureolysis. Particularly relevant in plants, ureases exert insecticidal and fungitoxic effects. Data on the jack bean urease and on jaburetox, a recombinant urease-derived peptide, have indicated that interactions with cell membrane lipids may be the basis of the non-enzymatic biological properties of ureases. Altogether, with this review we wanted to invite the readers to take a second look at ureases, very versatile proteins that happen also to catalyze the breakdown of urea into ammonia and carbamate.

Ureases: Historical aspects, catalytic, and non-catalytic properties – A

review

Karine Kappauna,b,1, Angela Regina Piovesana,c,1, Celia Regina Carlinia,b,⇑,2, Rodrigo Ligabue-Braunc,2

a Brain Institute (InsCer), Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6690, Prédio 63, Porto Alegre, RS CEP 90610-000, Brazil

b

Graduate Program in Medicine and Health Sciences, School of Medicine, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil

c

Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:

Received 7 March 2018

Revised 22 May 2018

Accepted 24 May 2018

Available online 28 May 2018

Keywords:

Virulence factor

Urea hydrolysis

Ureolytic microorganisms

Multifunctional proteins

Plant defense

Urease

a b s t r a c t

Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-containing enzyme produced by plants, fungi, and bacteria that catalyzes the hydrolysis of urea into ammonia and carbamate Urease is of historical impor-tance in Biochemistry as it was the first enzyme ever to be crystallized (1926) Finding nickel in urease’s active site (1975) was the first indication of a biological role for this metal In this review, historical and structural features, kinetics aspects, activation of the metallocenter and inhibitors of the urea hydrolyzing activity of ureases are discussed The review also deals with the non-enzymatic biological properties, whose discovery 40 years ago started a new chapter in the study of ureases Well recognized as virulence factors due to the production of ammonia and alkalinization in diseases by urease-positive microorgan-isms, ureases have pro-inflammatory, endocytosis-inducing and neurotoxic activities that do not require ureolysis Particularly relevant in plants, ureases exert insecticidal and fungitoxic effects Data on the jack bean urease and on jaburetox, a recombinant urease-derived peptide, have indicated that interactions with cell membrane lipids may be the basis of the non-enzymatic biological properties of ureases Altogether, with this review we wanted to invite the readers to take a second look at ureases, very ver-satile proteins that happen also to catalyze the breakdown of urea into ammonia and carbamate

Ó 2018 Production and hosting by Elsevier B.V on behalf of Cairo University This is an open access article

under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

History and molecular features of ureases Ureases (urea amidohydrolase, EC 3.5.1.5) are ubiquitous metalloenzymes, produced by plants, fungi and bacteria, but not

by animals The most proficient enzymes known to date, ureases

https://doi.org/10.1016/j.jare.2018.05.010

2090-1232/Ó 2018 Production and hosting by Elsevier B.V on behalf of Cairo University.

Peer review under responsibility of Cairo University.

⇑ Corresponding author.

E-mail address: celia.carlini@pucrs.br (C.R Carlini).

1

These authors contributed equally to this work.

2 These authors share the senior authorship.

Contents lists available atScienceDirect

Journal of Advanced Research

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catalyze the hydrolysis of urea into ammonia and carbamate

(which then decomposes into another ammonia molecule and

carbon dioxide), accelerating the rate of this reaction by a factor

of at least 1014when compared to the urea decomposition by

elim-ination reaction[1–4]

Computational modeling of urease proficiency led to the

pro-posal of a value up to 1032times the theoretical rate of uncatalyzed

urea hydrolysis[5] However, one can argue that, in solution, this

value is not realistic due to limits imposed by the diffusion of the

substrate in water

Urea, the natural substrate of ureases, was first isolated from

human urine by Rouelle in 1773 and about a half century later,

Wöhler achieved the synthesis of urea, the first organic molecule

to be obtained from inorganic ones[6] The first ureolytic

microor-ganism, Micrococcus ureae, was isolated by van Tiehem in 1864,

and the first enzyme with ureolytic activity was isolated from

putrid urine by Musculus in 1874 The name ‘‘urease” was

pro-posed in 1890 by Miquel[4] Urease contributed two historical

landmarks in Biochemistry First, the crystallization of urease

iso-lated from jack bean (Canavalia ensiformis) seeds by James B

Sum-ner, in 1926, demonstrated the proteinaceous nature of enzymes

[7], a discovery laureated with the Nobel Prize in Chemistry in

1946 Second, the biological significance of nickel was recognized

in 1975, after studies of Zerner’s group revealing the presence of

nickel ions in the active site of the jack bean urease (JBU),

obliga-tory for its catalytic activity[8] The identification of a plant toxin

as a urease in 2001 can be considered as a third breakthrough

involving ureases, as it led to the discovery of non-catalytic

prop-erties of these enzymes[9] This finding widened our knowledge

on the array of functions performed by these proteins, besides their

role in nitrogen metabolism[10]

Ureases are members of the superfamily of amidohydrolases

and phosphotriesterases, which display catalytically active metal

(s) in their active sites With a few exceptions reported [11,12],

ureases carry two Ni2+ions in their active sites[4,13] Ureases from different sources share about 55% identity in their primary sequences suggesting divergence from a common ancestral pro-tein X-ray crystallography studies revealed that plant and bacte-rial ureases share a common basic ‘‘trimeric” structure [4,14] The number of polypeptide chains that form the ‘‘monomer” or functional unit varies according to the source of urease For plant and fungal ureases this functional unit is a single polypeptide chain (a) The functional unit of bacterial ureases is formed by two sub-units (aandb, so far found only in the genus Helicobacter) or three (a,b andc) types of polypeptide chains The most abundant struc-ture of plant ureases is a dimer of trimers (a3)2 although a few dimeric/trimeric/tetrameric plant and also fungi ureases have been described Bacterial ureases are trimers ([abc]3) while Helicobacter pylori’s urease has been crystalized as a tetramer of trimers of dimers ([ab]3)4(reviewed in[10,14]) The amino acid sequences

of smaller subunits of prokaryotic ureases are collinear to the cor-responding region in the single chain of eukaryotic ureases[4]

Fig 1illustrates the structural features of ureases

The primitive state of these proteins – single- or three-chained –

is one of the unanswered questions regarding ureases Using phylogenetic inference and two algorithms applied to three differ-ent datasets, a 3-to-1 transition in the number of urease’s subunits was observed, implying a three-chained ancestral urease from which all the present enzymes derived In that scenario, the two-chained ureases in the genus Helicobacter are not evolutionary intermediates of the eukaryotic single-chained ureases[15]

Table 1presents an updated list of ureases for which molecular and kinetics characteristics are known

Activation and catalytic properties of ureases The active site of ureases consists, besides the two nickel atoms,

of one carbamylated lysine, four histidines and one aspartate

Fig 1 Urease structural conservation A functional unit can be formed by a heterotrimer (as in Sporosarcina pasteurii, PDB ID 2UBP ), a heterodimer (as in Helicobacter pylori, PDB ID 1E9Z ) or by a single unit (as in Canavalia ensiformis, PDB ID 3LA4 ) These functional units (or monomers) form larger complexes, such as trimers, hexamers or

Table 1

Biochemical and structural data on selected ureases of plants, bacteria and fungi.

Source

Isoform

GenBank identifier

Native M r Oligomeric state

Number of residues – M r subunit(s) a

for urea (mM)

Optimal pH 3D structure

(PDB ID)

Refs

PLANTS Arabidopsis thaliana

AT1G67550

Canavalia ensiformis

JBU

M65260.1

540 kDa

ɑ 6

840 aa 90.8 kDa

5.0–5.1 2.9–3.6 7.0–7.5 3LA4 [17–23]

Canavalia ensiformis

CNTX

180 kDa

ɑ 2

n.a.

95 kDa

Cajanus cajan

JN107804.1

540

kDa-ɑ 6

840 aa

90 kDa

Glycine max

Embryo-specific

AY230157

93.5 kDa

Glycine max

Ubiquitous

AY276866

345 kDa

a3

8.75

Morus alba

AB479106.1

175 kDa

ɑ 2

FUNGI Aspergillus nidulans 540 kDa

ɑ 6

840 aa

90 kDa

Aspergillus niger

XM_001388748.2

540 kDa

ɑ 6

837 aa

90 kDa

Cryptococcus gattii

CPC735_069440

180 kDa

ɑ 2

840 aa

90 kDa

Cryptococcus neoformans

CNAG_05540

90 kDa

Coccidioides posadasii

CPC735_069440

540

kDa-ɑ 6

840 aa

90 kDa

Coccidioides immitis

U81509

91.5 kDa

Schizosaccharomyces pombe a2 835 aa

91.2 kDa

Brevibacterium ammoniagenes 200 kDa

(ɑbc) 3

a

67 kDa

Brucella suis

Two operons

Helicobacter pylori

M60398

1.06 MDa ([ɑb] 3 )4

b

238 aa

30 kDa

a

569 aa

62 kDa

5.9 0.2–0.8 8.0–8.2 1E9Z [45–48]

Klebsiella aerogenes

M36068

(ɑbc) 3 c

100 aa 11.1 kDa b

106 aa 11.7 kDa

a

567 aa 60.3 kDa

Morganella morganii 590 kDa

(abc) 3

63 kDa

15 kDa

6 kDa

Providencia stuartii 230 kDa

(c2b2a) 2

c

9 kDa b

10 kDa

a

73

Proteus mirabilis

M31834

252 kDa (ɑbc) 3

c

100 aa

11 kDa b

109 aa 12.2 kDa

a

567 aa

residue The crystal structures of bacterial ureases from Klebsiella

aerogenes[50]and Sporosarcina (former Bacillus) pasteurii[56]first

revealed the architecture of the enzymes’ active site These two

ureases have nearly superimposable active sites, very similar to

those of other ureases characterized afterwards, implying that this

architecture is representative of all ureases In the active site, the

carbamylated lysine bridges the two nickel atoms, with Ni(1)

fur-ther coordinated by two histidines and Ni(2) by the ofur-ther two

his-tidines and by an aspartate residue Additionally, a hydroxide ion

bridges the two Ni atoms, which along with other three terminal

water molecules (W1, W2, W3), forms an H-bonded water

tetrahe-dral cluster in the active site (Fig 2)[4,14,2]

Besides the amino acid residues that compose the active site

itself other residues, including a conserved cysteine, form the

‘‘mobile flap”, which works as a gate for the substrate This flap

is composed by a helix-turn-helix motif and is responsible for

sub-strate influx and product efflux in ureases, especially via motion

control of a conserved histidine residue[2] In the catalysis, amino

acid residues of the mobile flap participate in the substrate

bind-ing, mainly through H bonds, thereby stabilizing the catalytic

tran-sition state and accelerating the reaction[2,4,14]

The mechanism for urea hydrolysis catalyzed by urease (Fig 2)

has been a hotly debated subject (see[64,65]) Currently, it seems

to be an agreement on the mechanism, strongly supported by

stud-ies with urease inhibitors [14,66–68] After taking the place of

water molecules W1-W3 (Fig 2A) in the urease active site, urea

binds to Ni(1) ion through the carbonyl oxygen, making the urea carbon more electrophilic and, thus, more susceptible to nucle-ophilic attack (Fig 2B) Then urea binds to Ni(2), through one of its amino nitrogen atoms, establishing a bidentate bond with urease (Fig 2C) This bond is believed to facilitate the water nucle-ophilic attack on the carbonyl carbon resulting in a tetrahedral intermediate (Fig 2D), from which NH3and carbamate are released (Fig 2E) The main controversy point was that while Benini et al.,

1999[65]proposed that the nucleophilic attack is performed by the bridging hydroxide which provides protons to the NH3group, Karplus et al., 1997[64]argued that it is a His residue from the active site mobile flap that acts as a general acid for this protonation As an alternative, Karplus et al., 1997[64]also consid-ered the monodentate binding of urea to Ni(1) with Ni(2) providing the water molecule as a nucleophile for the carbonyl carbon of urea

In addition to these two hypothesis, Estiu and Merz, 2007, based on simplified computer models for the active site, proposed that hydrolysis and elimination could occur competitively in ureases,

in which a ‘‘protein-assisted elimination” would be favored[69]

To achieve full ureolytic activity, the active site of ureases needs prior insertion of nickel ions and also carbamylation of its lysine residue In bacteria, four accessory proteins (UreD, UreF, UreG, and UreE) are involved in the assembly of urease’s active metallo-center For reviews on this topic see[13,70–73] In bacteria, the urease genes UreA, UreB, and UreC encoding the enzyme’s subunits are grouped with genes for the accessory proteins UreD, UreE, UreF,

Table 1 (continued)

Source

Isoform

GenBank identifier

Native M r Oligomeric state

Number of residues – M r subunit(s) a

for urea (mM)

Optimal pH 3D structure

(PDB ID)

Refs

61 kDa

Sporosarcina pasteurii

KR133628

260 kDa (ɑbc) 3

c

101 aa 11.1 kDa b

122 aa

14 kDa

a

570 aa 61.4 kDa

Staphylococcus leei 480 kDa

[(cba)5

c

12 kDa b

21 kDa

a

65 kDa

Staphylococcus saprophyticus 427 kDa

(cba) 4

c

13.9 kDa b 20.4 kDa

a

72.4 kDa

Staphylococcus xylosus

X74600

300 kDa (ɑbc) 3

c

16.3 kDa b 17.8 kDa

a

64 kDa

Ureaplasma ureolyticum

L40490

274 kDa (ɑbc) 3

c

102 aa 11.2 kDa b

121 aa 13.6 kDa

a

614 aa 66.6 kDa

5.0–5.2; 4.6 2.5 6.9–7.5 n.a [61–63]

a

Regardless of the names given to urease’s subunits in the initial or original reports, here the subunits were designated according to their homologous protein domains n.a not available

and UreG In the case of K aerogenes, these genes are organized in

an UreDABCEFG operon Knockout and complementation studies of

each accessory protein separately have shown that, UreE as an

exception, UreD, UreF and UreG are crucial for the production of a

fully activated ‘‘mature” urease[70,74,75]

The traditional model for urease activation starts with UreD, the

first protein that binds to the apo-urease oligomer, and serves as a

scaffold for the formation of the activation complex Then UreF

binds (UreABC–UreD)3, and acts as a GTPase-activating protein,

since its binding to (UreABC–UreDF)3 correlates to the GTPase

activity when further binding of UreG completes the activation

complex UreG, the first intrinsically disordered enzyme to be

described[76,77], acts as a GTPase delivering energy for the urease

maturation process As GTP is hydrolyzed, the nickel-binding

chap-erone UreE delivers the metal ions to the (UreABC–UreDFG)3

oligo-mer [76,77] This model has been further refined with the

increasing amount of structural information on individual urease

accessory proteins [14] In this new activation proposal, Ni2+

-bound UreE binds apo-UreG, facilitating GTP uptake by UreG

(pres-ence of Mg2+ions is required), with Ni2+ions being translocated

from UreE to UreG Then, the (UreDF)2 complex competes with

UreE for the Ni2+-UreG to form the supercomplex apo-urease/

Ni2+-(UreDFG)2 In the final step, KHCO3/NH4HCO3catalyzes GTP

hydrolysis by UreG, thus completing urease activation All urease

accessory proteins are taken as metallochaperones that bind and/

or transport nickel ions while driving the apo-urease into its fully

active conformation In plants and fungi, the functions of the

bacterial UreG and UreE chaperones appear to be combined in a single UreG protein, which carries a histidine-rich domain with metal binding properties in its N-terminal segment[78,79] The reason why eukaryotes lack UreE is still unknown[79]

The role of each accessory protein in the activation process has been a research hot topic in the last decade and there are some questions yet to be answered, mostly on the sequence of events and oligomerization state of each protein in the activation com-plex The description at low resolution by small-angle X-ray scat-tering of the K aerogenes (UreABC–UreD)3and (UreABC–UreDF)3 oligomers started to uncover what the activation complex looks like[80] Computational studies provided models of the activation complex[81] The crystal structure of H pylori’s UreD-UreF-UreG complex revealed the presence of tunnels that span the entire length of both UreF and UreD, through which the delivery of nickel ions from UreG to the apo-urease could possibly occur[73,82]

Ureases inhibitors Studies on urease’s inhibitors have been carried out both to pro-vide molecular insights on how the catalytic site machinery works

as well as searching for effective inhibitors to counterbalance urease’s catalyzed urea hydrolysis in a number of situations

[83,84] Urease inhibitors are a topic of intense investigation The substrate urea, urea analogues and ammonium ions (products of urea hydrolysis), are weak inhibitors of urease[4] Searching the

Fig 2 Catalytic mechanism of ureases Structure-based urease catalytic mechanism of the enzymatic hydrolysis of urea The Sporosarcina pasteurii urease residue-numbering scheme is used Please refer to the text for a stepwise description of the mechanism Note that Ni(1) and Ni(2) are labeled Ni1 and Ni2 in this figure Reproduced from Mazzei

et al [14] under permission from the Royal Chemical Society.

Web-of-Sciences database (March 6th, 2018) for articles with

‘‘urease” in the title retrieved 4509 documents, 920 were found

using ‘‘urease” and ‘‘inhibit⁄” of which 413 were published since

2010 Please refer to the next section, ‘‘Biological roles of ureases”,

for more information on the importance of ammonia release by

ureases

An extensive and detailed review on the different classes of

urease inhibitors can be found in[14] Other articles on this special

issue of Journal of Advanced Research deal in more details with

urease inhibitors

Sulfur compounds

Thiols, particularly b-mercaptoethanol, are of historic

importance as urease inhibitors that, back in 1980, provided to B

Zerner’s group crucial information on the active site of JBU[85]

Thiolate anions (R-S-) inhibit ureases in a competitive manner

X-ray analysis of S (B.) pasteurii urease complexed with

b-mercaptoethanol (PDB code 1UBP) revealed its thiolate anion

bridging the two Ni2+ions in the active site and the hydroxy group

further chelating the metallocenter [56] Sulfite also acts as

competitive pH-dependent inhibitor of urease[86]

Hydroxamic acids

Acetohydroxamic acid, the most studied derivative of this group

of metal-binding compounds, acts as a urease slow-binding

com-petitive inhibitor It has been found interacting with the two nickel

ions in the active sites of S.(B.) pasteurii (PDB code 4UBP), H pylori

(PDB code 1E9Y) and a mutated form of K aerogenes (PDB code

1FWE) ureases[86] So far, acetohydroxamic acid is the only urease

inhibitor with therapeutic application to treat hyperammonemia

in cirrhosis of H pylori positive-patients[87]and it has been used

to reduce urinary stones and treat urinary infections due to

P mirabilis infections [84,88] However, this compound induces

severe side effects, including teratogenesis, psychoneurological

and muscular symptoms[89], which limit its use and caused its

withdraw from the general market[84]

Phosphorous compounds

Amide and esters of phosphoric/thiophosphoric acids

Studies on phosphorus-based compounds as urease inhibitors

started in the 1970s after the observation that some

organophosphate-based insecticides inhibit soil urease [4,90] In

1980, Dixon et al described that phophoramidate inhibited JBU

through its binding to the two nickels in the enzyme’s active site

[65,85] Derivatives of phosphoric and thiophosphoric acid are

potent inhibitors of urease[4] A great number of derivatives have

been developed and patented for potential application in infections

by urease-producing pathogenic microorganisms[83]and in

agri-culture to avoid hydrolysis of urea used as fertilizer[84] For all the

derivatives of this class of inhibitors, the initial enzymatic

hydrol-ysis of the molecule generates diamidophosphate, which is

believed to be the actual urease inhibitor [14] The main issue

involving organophosphate inhibitors of urease is related to their

low stability in acidic pH To overcome this problem

non-hydrolysable aminophosphinic acids have been developed[91,92]

Phosphate

Phosphate is a pH-dependent urease competitive inhibitor in

the pH range 5.0–8.0, but negligible at pH higher than 7.5–8.0

[23,93] X-ray diffraction structural data on phosphate-inhibited

S.(B.) pasteurii urease inhibited with phosphate elucidated that

the binding mode involves the formation of four coordinated bonds

with both Ni ions in the enzyme’s molecule [93] It is a weak

inhibitor compared to its amides (phosphoramidates) that rank among the most active urease inhibitors

Fluoride The mode of inhibitory action of fluoride, explored mostly using S.(B.) pasteurii urease, was described as a pH-sensitive mixed inhi-bition, which varies from a weak competitive mode in acidic med-ium to a stronger uncompetitive mechanism in alkaline conditions

[57] Five crystal structures of the enzyme in its fluoride-inhibited state were analyzed to establish that one fluoride ion binds to Ni(1)

of the active site, while the nickel bridging hydroxide is replaced

by another fluoride ion[57]

Quinones Ubiquitous in the nature, quinones have bactericidal and anti-fungal activities, and participate of biologically relevant redox mechanisms Quinones were described as urease inhibitors in the 1970s in studies of Bremner’s group, pointing to 1,4-benzoquinone as a promising inhibitor of soil urease[94] More recently, Krajewska’s group reported on the kinetics of the inhibi-tion of JBU by quinones, demonstrating a general slow-binding concentration-dependent mechanism indicative of a covalent modification of the conserved cysteine residue in the mobile flap

of the active site In addition to the covalent modification, quinones might inhibit urease through arylation and oxidation of its thiol groups[95]

Polyphenols Catechol, the simplest molecule with a polyphenol scaffold, was shown to inhibit soil urease by Bremner and Douglas early in 1970s [94] Plants are rich sources of polyphenolic compounds with antioxidant and bactericidal properties, generally regarded

as beneficial for human health For instance, polyphenols present

in the green tea and other herbal beverages inhibited H pylori urease (HPU) in vitro and reduced infection by H pylori in Mongolian gerbils[96,97] The mechanism of inhibition of urease

by catechol is not yet fully understood Current hypothesis are that inhibition by catechols could be due to a time dependent oxidation

to ortho-benzoquinone which acts as the actual inhibitor by mod-ifying protein’s functional groups[98,99], and/or that polyphenols can coordinate with transition metals forming catechol–metal complexes, inactivating urease’s metallocenter[100]

Other urease inhibitors Although in most cases detailed structural data are not avail-able, other classes of urease inhibitors are known, including boron-containing acids, citrates, and heavy metals For a review

on these topics see[4,14] Heavy metals such as Hg, Ag, and Cu are slow reacting inhibitors of ureases[101,102] Bismuth (Bi3+) was shown to inactivate HPU by interacting with the cysteine resi-due of the mobile flap[103] Due to the bactericidal activity, bis-muth compounds have been widely used to treat gastric ulcers associated to H pylori infection[104,105]

Biological roles of ureases that require ureolytic activity Urease activity enables microorganisms to use urea as their sole nitrogen source Urease synthesis may be constitutive or synthe-sized as a stress-related response of bacteria to counteract low environmental pH [106] Ureolytic activity of the human gut microbiota hydrolyzes up to 30% of all urea produced in our bodies

[107] Microbial ureases are important also in dental health[108].

The production of alkali subsequent to salivary urea cleavage by

oral microbiota urease was shown to inhibit dental cavities and

plaque formation [109] In ruminants, animal-derived urea is

cleaved by bacterial ureases in the forestomach, releasing

ammo-nia as nitrogen source for the rumen microbiota, which in turn

serves as biomass to feed the animals[110,111]

Pathogenesis of many clinical conditions in humans and other

animals are related directly to the ureolytic activity of bacterial

or fungal enzymes [112,113] Some examples are as follows

Proteus mirabilis is the most common organism that causes urinary

stones in humans, due to urine alkalization promoted by its urease,

contributing to the pathogenesis of pyelonephritis and catheter

encrustation Precipitation of urinary salts in the alkalinized urine

results in struvite and carbonate apatite crystallization[114] The

bacterium H pylori colonizes the stomach mucosa of half of the

world’s population, significantly increasing the risk of gastric

ulcers and cancer [113,115,116] HPU, which constitutes about

10% of the total cell protein, enables bacterial survival in the

stom-ach by neutralizing the acidic medium[117] Ureolytic organisms

in the digestive or urinary tract potentially contribute to hepatic

encephalopathy and coma resulting in hyperammonemia and

brain intoxication[118] Reduction of the ureolytic bacteria load

and the use of acetohydroxamic acid as a urease inhibitor are

con-sidered therapeutic approaches under these conditions[119–121]

Other pathogens also produce urease to acquire acid resistance and

enable colonization, among which are Shiga-toxin producing

Escherichia coli[122], Yersinia enterocolitica[123], K pneumoniae

[124], Brucella abortus [125], and Haemophilus influenza [126]

Fungal ureases are involved in the pathogenesis of human

crypto-coccosis by Cryptococcus neoformans[127,128], and Cryptococcus

gattii [35], and of coccidiodomycosis (San Joaquin Valley fever)

by Coccidioides immitis and C posadasii[37] However, the role of

microbial ureases as virulence factors has a still largely ignored

contribution of non-enzymatic properties of these proteins, a

sub-ject that will be covered in the following section

Urease is ubiquitous in plants and can be found in all vegetal

tissues[129,130] Nitrogen is a limiting element for plant growth,

second only to carbon Worldwide used as a soil fertilizer, urea is a

relevant N source for plants, and dedicated urea transporters

actively import this compound from the soil[131] Urea hydrolysis

to release ammonia and carbon dioxide is the main physiological

role attributed to ureases in plants[130,132] Urease is abundant

in the soil, both in living bacteria and as extracellular urease,

bound to clays and humic substances [133,134] Ureolysis by

cell-free ureases alkalinizes the soil inducing calcium carbonate

precipitation and affecting the availability of minerals[135,136]

In addition to that, high levels of soil urease reduce the efficiency

of urea fertilization leading to loss of ammonia into the

atmo-sphere and ammonia-induced phytotoxicity[90,137] The search

for urease inhibitors with agricultural applicability to optimize

urea fertilization is an intense field of investigation These topics

are broadly covered in other articles of this thematic issue of the

Journal of Advanced Research

Biological properties of ureases independent of ureolysis

Table 2lists the biological properties of ureases found not to

require ureolysis, either because urea is not available or its

concen-tration is negligible, or the study employed ureases that were

enzymatically incompetent (either with blocked active sites or in

the inactive, nickel-deprived, apo-urease form)

Ureases play a role in cell-to-cell or organism-to-organism

communication Arginases with lectin properties from the lichens

Evernia prunastri and Xanthoria parietina were shown to bind to a

glycosylated urease in the cell wall of the homologous algae The polygalactosylated urease is produced only in the season when the algal cells divide assuring recognition of the phycobiont by its fungal partner in the mutualistic association of these lichens

[138,139] Ureases were evaluated for a role in soybean nodulation by the diazotrophic bacterium Bradyrhizobium japonicum[140] Soybean and jack bean ureases were characterized as chemotactic factors recognized by the bacterial cells in vitro Independent of the urease status of the nodulating bacteria, urease-deficient mutant soybean plants had fewer but larger nodules when compared to the wild-type plant Leghemoglobin production in wild-wild-type plants was higher and peaked earlier than in urease-deficient plants, indicat-ing a less efficient process of nitrogen fixation Inhibition of urease activity in wild-type plants did not reproduce the results seen in mutated plants These data made clear that soybean urease(s), but not the bacterial enzyme, participate(s) somehow of the plant-diazotrophic bacteria symbiosis This role of the soybean urease does not require ureolysis and is relevant for biological nitrogen fixation by the plant[140]

Among microbial ureases that play a role as virulence factors, much attention is given to HPU because of its crucial role in the pathogenesis of gastric diseases Production of urease proved to

be essential to allow stomach colonization by H pylori, however studies carried out in the early 1990s have shown that neutraliza-tion of gastric acidity is not the only funcneutraliza-tion of the protein

[141,142] Following the steps of our previous observations made

on ureases from jack bean (C ensiformis) and from S.(B.) pasteurii (reviewed in[10]– see next sections), we have reported several other biological properties of the purified recombinant HPU, observed in the 10 6–10 8M range of protein concentration These properties include induction of lipoxygenase-dependent activation and aggregation of rabbit[143]and human platelets[144]; induc-tion of lipoxygenase-dependent chemotaxis and ROS producinduc-tion in human neutrophils [145]; delaying apoptosis in human neu-trophils[145]and in gastric epithelial cells[146]; increase of the lipoxygenase content in neutrophils[145]; induction in platelets

of the production of lipoxygenase-derived eicosanoids[143]; pro-motion of angiogenesis in human umbilical endothelial cells and

in the chicken embryo chorioallantoic membrane model [146]; and induction of processing of pre-mRNA encoding pro-inflammatory cytokines in human platelets [144] Most of these effects are also displayed by an enzyme-inhibited HPU, while some are induced by one of its isolated subunits alone[144], indicating that these biological effects do not require urea hydrolysis Other groups also reported biological roles of HPU that are carried out

by one of its subunits, implying absence of ureolysis HPU’s subunit

B was shown to bind to Th17 lymphocytes[147]and to CD74 on gastric epithelial cells thereby eliciting production of IL-8 [148] HPU’s subunit A contains a nuclear localization signal (sequence

21KKRKEK26), and it was found in the nuclei of COS-7 cells

[149,150]and AGS gastric epithelial cells, inducing alterations in the cells’ morphology[150]

Altogether these non-enzymatic biological effects of HPU point out to a relevant contribution (yet mostly ignored) of this protein

to the inflammatory process that underlies the gastric diseases caused by H pylori Because HPU activates non-gastric cells such

as platelets, neutrophils, endothelial cells, among others, it may contribute as well to the pathogenesis of extragastric illnesses, in particular cardiovascular diseases Probably none of the future urease inhibitors that are being conceived or are presently under development will have any use to counteract HPU’s pro-inflammatory effects or other unwanted contributions of this protein that are not due to its ureolytic activity Thus, there is an urgent need to understand the structural basis of the non-enzymatic biological properties of HPU, and of other microbial

ureases with relevant roles as virulence factors, aiming the design

of drugs that could specifically block these other activities Such

new urease inhibitors could be used alone or together with

ureol-ysis inhibitors, to target all the noxious effect of ureases involved in

pathogenesis

Neurotoxicity of ureases

The discovery of the non-enzymatic properties of ureases is

clo-sely related to the study of their neurotoxicity, both in rodents and

in insects Canatoxin (CNTX) is an isoform of C ensiformis urease,

first isolated from the plant seeds as a neurotoxic protein causing

convulsions and death of rats and mice, with an LD50 2 mg/kg,

given by intraperitoneal route[151] Two decades after the

isola-tion of CNTX, it became evident that the neurotoxic protein is

actu-ally an isoform of the most abundant urease (JBU) found in the

same seeds[9] Canatoxin is a non-covalent dimer of95 kDa

sub-units with one zinc and one nickel atom per subunit[9,12]what

probably explains its lower ureolytic activity CNTX and JBU differ

in one order of magnitude in their sensitivity to the irreversible

inhibitor p-hydroxy-mercurybenzoate (pHMB), an oxidant of thiol

groups[9]and in their metal-binding affinities[152]

Studies on CNTX have indicated that its primary mechanism of

action at the cellular level is to induce exocytosis, triggering a

sig-naling pathway that characteristically involves eicosanoids derived

from the lipoxygenases pathway (reviewed in[10]) This biological

property of CNTX was reported in a number of mammalian models,

both in vivo and in vitro, among which are blood platelets and rat

brain synaptosomes The aggregating activity of CNTX in rabbit,

rat, guinea pig or human platelets occurs in the nanomolar range

[153] CNTX-activated platelets recruit a lipoxygenase-mediated

pathway that leads to influx of external Ca2+through opening of

voltage-gated Ca2+ channels and without release of intracellular

[Ca2+] pools The increased cytoplasmic [Ca2+] triggers exocytosis

of platelet granules that contain ADP, which in turn induces the

aggregation response[153,154] Later the ability to induce platelet

aggregation was reported for JBU [9], the embryo-specific[155]

and the ubiquitous [156] isoforms of soybean ureases, B.(S.)

pasteurii urease [155,157], and HPU [143], thus it is a property

common to one-, two-, and three-chained ureases

The observations that pHMB-treated CNTX, in which the

ure-olytic activity is irreversibly blocked, was still lethal to mice and

still able to promote platelet aggregation set the ground for the

dis-covery of the non-enzymatic biological properties of ureases[9] In

the following two decades, a lot more of ureolysis-unrelated effects were described for C ensiformis ureases as well as for ureases from other sources (reviewed in[10])

The exocytosis inducing effect of CNTX was later characterized

in rat brain synaptosomes, which responded dose-dependently to the neurotoxin by releasing neurotransmitter vesicles previously loaded with radiolabeled serotonin or dopamine At 500 nM CNTX, the amount of neurotransmitter released from the synaptosomes was similar to that obtained by depolarization with 50 mM KCl

[158] The ability of CNTX to promote secretion in synaptosomes correlates with the neurotoxicity it induces in vivo in mice and rats The medullar origin of CNTX-induced seizures and other CNS-related effects were described in rodents[159]

More recent data have shown that JBU (10–100 nM) induces

Ca2+ events in cultured rat hippocampal neurons, an effect also observed for HPU (Piovesan, A.R., unpublished results) In patch clamp experiments, it was observed that JBU increases the fre-quency of spontaneous firing action potentials in cultured rat hip-pocampus neurons, rising the amplitude of sodium currents, and apparently not affecting potassium currents A higher frequency

of spontaneous excitatory post synaptic currents was also seen, consistent with a seizure-like activity (Dal Belo, C A., unpublished data) Studies using microPET (Positron Emission Tomography) indicated an increase of30% in the uptake of 18

Fluor-desoxy-glucose in the brain of CNTX-treated anaesthetized rats, particularly affecting the hippocampus, a typical finding for seizure-inducing drugs (De Almeida, C.G.M., unpublished results) Similar to our observations, JBU had been previously reported to

be lethal and to produce seizures in mice and rabbits after intra-venous administration[160] Likewise, purified HPU was shown

to kill mice upon intraperitoneal injection, producing hypothermia, convulsions and death[161] In both studies, the neurotoxicity of the ureases was attributed to the high levels of ammonia found

in the animal’s blood Although hyperammonemia probably con-tributes to the neurotoxic effects induced by CNTX in mice and rats, surely it does not tell the whole story, considering that pHMB-treated CNTX still caused neurotoxic symptoms and sei-zures leading to death of the animals[9]

Contributions of ureases to plant defense against predators and pathogens

The first description of the insecticidal effect of a urease was published in 1997 showing that ingestion of CNTX killed insects

Table 2

Ureolysis-independent biological properties of selected ureases and urease-derived peptides.

Ureases and

derived-peptide

Entomotoxic properties

Antifungal activity

Mammal neurotoxicity

Exocytosis in platelets

Eicosanoid signaling

Chemotactic activity PLANTS

BACTERIA

UREASE-DERIVED PEPTIDES

CNTX, canatoxin (C ensiformis); JBU, jackbean urease (C ensiformis); eSBU, embryo-specific soybean urease (G max); uSBU, ubiquitous soybean urease; GHU, Gossypium hirsutum (cotton) urease; PPU, pipeon pig urease (C cajan); SPU, S pasteurii urease; HPU, H pylori urease; PMU, P mirabilis urease; BJU, B japonicum urease; JBTX, jaburetox; SYTX, soyuretox.

U presence of biological activity; ✗ absence of biological activity; ** Recombinant protein; n.d not determined; # unpublished result.

[162] The susceptibility of the insects to CNTX’s lethal effect

depended on the type of their digestive enzymes Insects with

acidic midguts and cathepsin-like proteinases, like the cowpea

weaver Callosobruchus maculatus (Bruchidae) and the kissing bug

Rhodnius prolixus (Hemiptera), were susceptible to CNTX while

insects with alkaline midguts and trypsin-like enzymes were not

These data were interpreted as evidence for the need of proteolytic

activation of CNTX that, once ingested, is hydrolyzed by insect

cathepsin-like enzyme(s) releasing an internal peptide(s) with

insecticidal activity In fact preventing CNTX hydrolysis by adding

a cathepsin B inhibitor simultaneously with the toxin in the

insects’ diet protected them against the lethal effect[162] In the

following years we described that JBU/CNTX and the embryo

speci-fic soybean urease were insecticidal against the hemipterans

Nezara viridula[163], Dysdercus peruvianus [155,164], Oncopeltus

fasciatus [165], and K Ponnuraj’s group in India reported the

insecticidal effect of the pigeon pea urease (Cajanus cajan) against

Callosobruchus chinensis[25]

The proteolytic activation of CNTX by insect cathepsin-like

enzymes was further investigated Insecticidal peptides were

iso-lated from CNTX’s fragments after digestion with C maculatus

enzymes[166] The most active peptide, pepcanatox, with a

molec-ular mass of10 kDa had its N-terminal sequence determined and,

based on this information, a recombinant peptide named jaburetox

was obtained by heterologous expression in E coli[167] Cathepsin

D-like enzymes from D peruvianus midgut that were able to

per-form hydrolysis of CNTX/JBU and release the insecticidal peptide

were characterized[164,168,169] A similar study was performed

with JBU and the milkweed bug Oncopeltus fasciatus, identifying a

cathepsin L that hydrolyzed the urease to release a10 kDa

ento-motoxic peptide[165]

The recombinant peptide jaburetox was cloned using as

tem-plate the cDNA of JBURE-II, a third isoform of urease found in C

ensiformis[170,171] Based on jaburetox’s sequence, a recombinant

insecticidal peptide called soyuretox was produced[172]having as

template the cDNA of the ubiquitous soybean urease which, like

the embryo-specific urease, also kills R prolixus [156]

Interest-ingly, the region that encompasses the jaburetox/soyuretox

sequence, comprising about 90 amino acid residues, displays a

lower similarity when compared to that of the complete sequence

of different ureases, suggesting less evolutionary pressure to

con-serve this entomotoxic ‘‘domain” of plant ureases[15,167]

But the proteolytic release of entomotoxic peptides does not tell

the whole story of urease’s entomotoxicity Evidences showing

that the entire urease molecule is entomotoxic per se started to

add up with studies on the anti-diuretic effect of C ensiformis

ureases In Carlini et al., [162], we showed that CNTX produced

an important anti-diuretic effect in R prolixus that peaked about

4 h after the insects received the ‘‘meal” containing the toxin,

dis-appearing after 24 h However, the hydrolysis of CNTX in the insect

midgut was not detected before 18 h, suggesting that the

anti-diuretic effect was produced by the entire protein Later, JBU and

the jaburetox peptide were shown to cause anti-diuresis in R

pro-lixus’ isolated Malpighian tubules in the concentration range of

10 10and 10 15M, respectively[173] Surprisingly, although both

molecules induced antidiuretic effects, JBU and jaburetox triggered

different signaling pathways leading to antidiuresis[173] In the

following years other papers were published by our group

describ-ing a list of entomotoxic effects of JBU, some of which are not

shared with jaburetox, such as alteration in water transport and

of the contractility in the crop of R prolixus[174] Similar to the

data indicating recruitment by ureases of eicosanoid-mediated

pathways in mammalian systems (reviewed in[10]), JBU effects

in insects required a phospholipase A2type XII[175]and

prosta-glandins [176] JBU and jaburetox targeted the immune system

of R prolixus, inducing an eicosanoid-dependent aggregation of

hemocytes and alterations in cell morphology[176,177]that ren-der the insect more susceptible to entomopathogenic bacteria

[177] Both JBU and jaburetox are neurotoxic to insects from different orders Jaburetox was immunolocalized in the brain of Triatoma infestans (Hemiptera) and neurotoxic symptoms preceded death

of the insects injected with the peptide[178] JBU-induced effects were studied in the cockroach Nauphoeta cinerea (Blatodea) reveal-ing that both, the central and the peripheral nervous systems are targeted by the urease, with alterations of the cholinergic, octopaminergic and GABAergic pathways as part of its entomo-toxic mode of action [179] The effects of JBU were also investigated on neuromuscular junctions of Locusta migratoria (Orthoptera) and of Drosophila melanogaster (Diptera), and the resulting data pointed to interference of JBU on neurotransmitter release, probably by disruption of the calcium machinery in the pre-synaptic region of insect neurons[180]

Previous studies with B.(S.) pasteurii urease suggested lack of insecticidal properties for microbial ureases, which was attributed

to the absence of part of jaburetox’s sequence in those proteins

[155] However, later reports on insecticidal activity of ureases of bacteria from Photorhabdus and Xenorhabdus genera[181], Yersinia pseudotuberculosis [182] and P mirabilis (Broll, V et al., unpublished results) indicated that bacterial ureases are indeed entomotoxic and insecticidal, in agreement to the fact that ureases contain other entomotoxic domains besides the sequence corre-sponding to jaburetox

Ureases are toxic against filamentous fungi and yeasts [183] The fungitoxic activity of CNTX was the first reported showing that the protein at 2% concentration caused growth inhibition of the phytopathogenic filamentous fungi Macrophomina phaseolina, Colletotrichum gloesporioides and Sclerotium rolfsii [184] Becker-Ritt et al., 2007, reported that JBU and the soybean embryo-specific ureases inhibited growth and/or spore germina-tion of seven other species of filamentous fungi at sub-micromolar concentrations and caused damage to cell wall, even after blockage of their ureolytic active sites In this same study, the two-chained HPU also inhibited fungal growth although with less efficiency [185] The native ureases of cotton seeds (G hirsutum) [30]and of pigeon pea [25], and the recombinant non-ureolytic apoureases, JBURE-IIb[171] and a ubiquitous soy-bean urease fused to glutathione transferase [156], were also shown to be detrimental to filamentous fungi

In Postal et al., 2012, JBU was tested in the 10 6–10 7M range against different yeast species and caused inhibition of prolifera-tion and of glucose metabolism, morphological alteraprolifera-tions with pseudohyphae formation, and cell membrane permeabilization, eventually leading to cell death[186] Jaburetox induced similar effects against the yeasts but at one to two orders of magnitude higher doses Studies with peptides from a papain-hydrolyzed JBU indicated the presence of other fungitoxic domains in the pro-tein, besides jaburetox[186] Soyuretox, a peptide derived from the soybean ubiquitous urease, is also fungitoxic in the same con-centration range as observed for jaburetox[172] Detached leaves

of ‘‘urease-null” soybean transgenic plants, due to co-suppression

of ureases genes, and infected with uredospores of the Asian rust fungus Phakopsora pachyrhizi developed more lesions and pustules when compared to leaves of wild plants with normal levels of ureases, suggesting a protective role of ureases against fungal dis-eases in the wild plants[187]

Interestingly, a non-catalytical urease was identified in the soy-bean genome This urease lacks critical features of the enzyme’s active site, but it is expressed in various plant tissues[188], rein-forcing the multifunctional characteristics of the protein, especially when related to plant defense It is tempting to predict that more

of these non-catalytical ureases will be found as more plant

genomes are decoded Altogether these data suggests that

urease-overexpressing plants or transgenic plants jaburetox/soyuretox

may represent alternatives to achieve resistance to insect

her-bivory and/or fungal disease in agriculture In this context it is

important to mention that ureases can be generally regarded as

biosafe proteins, which are present in relatively large quantities

in most edible plants and are particularly abundant in seeds of

legumes and in fruits such as tomatoes, melon, and watermelon,

that are eaten in raw state[129,132] Although more studies are

needed to ascertain the biosafety of urease-derived peptides, no

acute toxicity was detected for jaburetox given in high doses either

injected or by oral route to mice and neonate rats [167]

Preliminary data obtained for soyuretox in the zebrafish (Danio

rerio) model indicated toxicity only in the highest tested doses

(Kappaun, K et al., unpublished results)

Structural aspects of jaburetox

Models of the tridimensional structure of jaburetox[167,189]

indicated the existence in the C-terminal half of the peptide of a

prominent b-hairpin motif, a feature that could be related to a

pore-forming activity eventually leading its neurotoxicity A

b-hairpin in the region of JBU corresponding to jaburetox was found

in its crystallographic structure[22] Aiming to carry out structure

versus activity studies on jaburetox, three mutants corresponding

to truncated versions of the peptide were obtained: Jbtx D b,

which lacked the b-hairpin motif (residues 61–74 deleted); Jbtx

N-ter (residues 1–44), corresponding to the N-terminal half; and

Jbtx C-ter (residues 45–93), corresponding to the C-terminal half

of jaburetox[190] In insect bioassays, the JbtxD b peptide kept

the entomotoxic properties of the whole peptide, clearly indicating

that theb-hairpin motif is not required for the insecticidal effect

On the other hand, while Jbtx N-ter remained entomotoxic, the

Jbtx C-ter peptide, which contains the b-hairpin motif, was less

active or inactive when tested on two different insect models

The data support the conclusion that the N-terminal half of

jabure-tox carries its most important entomojabure-toxic domain[190]

Molecular dynamics studies employing long simulations of

jaburetox in aqueous medium suggested that the peptide becomes

largely unstructured after 500 ns, more accentuated in its

N-terminal domain, while the initial structure observed for its

moi-ety in JBU’s crystals is completely lost [190] Subsequently light

scattering, circular dichroism and nuclear magnetic resonance

spectroscopy studies of jaburetox in solution determined that it

is an intrinsically disordered polypeptide[191] Regions of

jabure-tox which exhibited tendency to form one small alpha-helix close

to the N terminus, and two turn-like motifs, in the central portion

and close to the C terminus, respectively, were predicted as sites of

potential interaction with other proteins or lipids, suggesting that

upon such interactions structural changes could be triggered to

drive the peptide into a biologically active conformation [191]

The solution structure of soyuretox was determined using the

same methodologies and revealed its intrinsically disordered

nature, although with more secondary structure elements when

compared to jaburetox (Kappaun, K et al., unpublished results)

Interaction of ureases and urease-derived peptides with lipids

and membranes

The interaction of jaburetox with lipid membranes was first

reported by Barros et al., 2009[189] In this study, jaburetox was

shown to cause leakage of carboxyfluorescein entrapped inside

large unilamellar vesicles, without lysis of the liposomes The

leak-age was greater in vesicles composed by acidic lipids and

depended on the state of aggregation of jaburetox Molecular

dynamics applied to jaburetox suggested that itsb-hairpin motif could anchor at polar/non-polar interfaces [189] However, as mentioned earlier, even if theb-hairpin does interact with insect membranes, it is not essential for the entomotoxic properties of jaburetox Moreover all three truncated versions of jaburetox developed by Martinelli et al., 2014, disrupted liposomes, revealing the presence of more than one lipid interacting domain in the pep-tide[190]

In another study, JBU, jaburetox and its mutated peptides were tested for an ion channel forming activity in planar lipid bilayers

[192] All proteins formed well resolved, highly cation-selective channels exhibiting two conducting states (7–18 pS and 32–79

pS, respectively) Urease (20 nM) and Jbtx N-ter (1lM) were more active at negative potentials, while the channels formed by the other peptides were not voltage-dependent This study was the first direct demonstration of the capacity of C ensiformis urease and jaburetox to permeabilize membranes through an ion channel-based mechanism, which may be the basis of their diverse biological activities Molecular models of JBU showed that the moi-ety corresponding to jaburetox is well exposed at the protein’s sur-face, from where it can probably ‘‘enforce” the interaction of the entire urease with lipid bilayers, a hypothesis formulated to explain why the polypeptides share many, although not identical, biological properties[192]

To elucidate whether an interaction with lipids could induce conformational changes in the intrinsically disordered molecule

of jaburetox, the structural behavior of the peptide was probed using nuclear magnetic resonance and circular dichroism spectro-scopies when in contact with membranes models[193] The inter-action of jaburetox with SDS micelles increased its content of secondary and tertiary structure elements When exposed to large unilamellar vesicles and bicelles prepared with phospholipids, con-formational changes were observed mostly in N-terminal regions, but without significant acquisition of secondary structure motifs Fluorescence microscopy was used to demonstrate that the lipid vesicles could displace the interaction of jaburetox with lipid-rich membranes of the cockroach nervous chord These data sug-gested that contacts of the N-terminal moiety of jaburetox with membrane phospholipids lead to its anchorage to cell membranes and promote conformation changes of jaburetox into a more ordered structure that could facilitate its interaction with membrane-bound target proteins[193]

Further studies aiming to elucidate the mechanism of interac-tion of JBU and jaburetox with lipid membranes were carried out using multilamellar liposomes with a lipid composition simulating that of human platelets, subjected to dynamic light scattering and small angle X-rays scattering (SAXS) analyses[194] Results were obtained indicating that both JBU and jaburetox are able to insert themselves into the lipid bilayers, reducing the hydrodynamic radius of the vesicles, altering the lamellar repeat distance, the number of lamellae, and decreasing the membrane’s fluidity The interaction of jaburetox affected the vesicle’s internal bilayers and caused more drastic effect on the multilamellar organization

of the liposomes than did JBU In the same study, the interaction

of JBU with giant unilamellar vesicles (GUVs) made of fluorescent phospholipids showed that JBU caused membrane perturbation with formation of tethers The data reinforced the idea that JBU can interact with multilamellar liposomes, probably by inserting its jaburetox ‘‘domain” into the vesicle’s external membrane[194]

Conclusions and future perspectives While the history of research on urease as an enzyme is almost

150 years old, dating back to the 1870s, the knowledge that ureases perform other biological roles unrelated to ureolysis is